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. 2012 Feb 22;32(8):2877–2885. doi: 10.1523/JNEUROSCI.3360-11.2012

Figure 1.

Figure 1.

Comparison of complexin-1 and complexin-3 sequences and analysis of the C-terminal complexin-1 sequence in clamping spontaneous mini exocytosis. A, Alignment of the mouse complexin-1 and complexin-3 sequences. Identical residues are highlighted in yellow and similar residues in green. The positions of the accessory and the SNARE-binding central α-helices are marked in blue and red, respectively, and the C-terminal isoprenylation sequence in complexin-3 is underlined. The positions in the complexin sequences at which the complexin-1 and complexin-3 sequences are swapped to generate complexin-1/3 hybrids (see Fig. 4) or where complexin-1 was truncated are marked by arrows, and the residues that were subjected to point mutations (see Fig. 7) are identified by asterisks. B, Immunoblots of cultured neurons that were infected either with control lentivirus (Control), or with lentivirus expressing the complexin-1/2 shRNA alone (Cpx KD) or together with various complexin rescue proteins as indicated [Cpx1WT, wild-type complexin-1; various complexin-1 point mutants (Cpx1L117K; Cpx1L117W; Cpx1I101W; Cpx1V120P); Cpx11–86, C-terminally truncated complexin-1; Cpx3, wild-type complexin-3; Cpx1–Cpx3 and Cpx3–Cpx1, complexin-1/3 and complexin-3/1 hybrid proteins; see A]. Neurons were infected at DIV5 and analyzed at DIV14. Although the blots indicate robust expression of all complexin proteins analyzed, expression levels cannot be directly compared between proteins because different complexin proteins exhibit distinct antibody reactivities. The top complexin blot was probed with the complexin antibody P942 (see Materials and Methods), the blot analyzing truncated complexin-11–86 was probed with antibody L669, and the complexin-3 and complexin-1/3 swap mutants were probed with antibody 122 301. Cpx3–Cpx1 was detected both with antibodies P942 and 122 301. C, Representative images of cultured cortical neurons infected with control lentivirus or with lentivirus expressing the complexin shRNA plus either GFP or wild-type complexin-1 (Cpx1). Neurons were infected at DIV4, fixed at DIV14, and stained by double-immunofluorescence labeling with the polyclonal complexin antibody P942 and monoclonal antibodies to either MAP2 (left) or synaptophysin (right) antibodies. Scale bar in the right bottom corner applies to all images in a set.